The invention relates to a method of two-dimensional heteronuclear correlation spectroscopy for the investigation of solid state samples containing a first type of nucleus (.sup.1 H) and a second type of nucleus (.sup.13 C), in a nuclear magnetic resonance spectrometer by means of a pulse sequence of nuclear magnetic resonance which pulse sequence comprises a preparation interval, an evolution interval, a mixing interval and a detection interval, wherein during the preparation interval the first type of nucleus is excited by means of at least one preparation radio frequency pulse in a first frequency band, and is exposed to evolution radio frequency pulses in the first frequency band during the evolution interval, wherein during the mixing interval the first type of nucleus is irradiated with cross polarisation radio frequency pulses in the first frequency band for transfer of nuclear magnetization to the second type of nucleus, and the second type of nucleus is irradiated in a second frequency band with at least one excitation radio frequency pulse, and wherein during the detection interval the first type of nucleus is exposed to at least one decoupling radio frequency pulse in the first frequency band, whereas a free induction decay of the second type of nucleus is detected in the second frequency band and wherein the pulse sequence (1.ltoreq.p.ltoreq.n) is carried out n times in succession with identical preparation interval, mixing interval and detection interval, however with altered evolution interval, and wherein the sample rotates at a rotational frequency of more than 1 kHz about an axis which is tilted by approximately 54.degree. with respect to the axis of a homogeneous magnetic field.
A method of this type is known from EP-A 0 481 256 (U.S. Pat. No. 5,117,186).
Nuclear magnetic resonance (NMR) is a phenomenon which occurs in connection with a selected group of atomic nuclei and is based on the existence of nuclear magnetic moments in these atomic nuclei. If an atomic nucleus having a nuclear spin is introduced into a strong uniform and static magnetic field (a so-called "Zeeman field") and is excited by means of a weak radio frequency (RF) magnetic field, the nuclear spin precesses at a natural resonance frequency, the Larmor frequency, which is characteristic for each type of nucleus with a nuclear spin, and depends on the strength of the magnetic field prevailing at the location of the nucleus. Typical atomic nuclei with a magnetic moment are e.g. .sup.1 H, .sup.13 C, .sup.19 F and .sup.31 P. The resonance frequencies of the nuclei can be observed by observing the transverse magnetization which occurs after a strong RF pulse. It is common practice to convert the measured signal into a frequency spectrum by means of Fourier transformation.
Although identical nuclei have the same frequency dependence on the magnetic field, differences in the direct chemical environment of each nucleus can modify the magnetic field such that nuclei of the same sample do not experience the same effective magnetic field. The differences in the local magnetic field cause spectral shifts of the Larmor frequency between two nuclei of this type which are chemically not equivalent, which are called "chemical shifts". These chemical shifts are interesting since they provide information about the number and the position of the atoms in a molecule and about the relative arrangement of neighbouring molecules within a compound.
Unfortunately, it is not always possible to interpret the frequency spectra caused by the chemical shifts since there are still other interactions which might be dominant.
This applies in particular to NMR spectroscopy of solid samples. In NMR spectroscopy of fluids, the fast movement of the molecules has the tendency to isolate the nuclei and separate the nuclear interactions such that it is much easier to recognize different nuclei in the spectrum. In solid state NMR there are numerous interactions between the molecules which obscure the result. The magnetic moments e.g. of neighbouring nuclei disturb each other leading to interactions which are called "dipole-dipole couplings". These couplings widen the characteristic resonance lines and cover the "fine" resonance structure caused by the chemical shift. A further problem occurring in connection with solid samples and not with fluids consists in that the orientation of the molecules in solids in relation to the Zeeman field applied, is relatively fixed and, for that reason, the chemical shifts are anisotropic such that a contribution to the resonance frequency depends on the spatial orientation of the molecules relative to the magnetic field applied. Consequently, it is essential to suppress some of these interactions in order to achieve an informative result for the others. This is usually achieved by exciting the system at selected frequencies so that undesired interactions are removed or at least averaged to a reduced amplitude. In solids e.g. the above-mentioned anisotropy of the chemical shift is usually largely reduced by orienting the solid sample at the so-called "magic angle" (54.degree.44') with respect to the magnetic field applied and rotating it at this angle at a relatively fast frequency, whereby the anisotropic field components are averaged to zero.
In a similar manner it is possible by means of known techniques to reduce the undesired spin-spin interactions by irradiating the nuclei with additional RF pulses at or near the Larmor frequencies. By carefully selecting different polarizations and phases of the RF pulses, the magnetization of the disturbing nuclear spin systems in neighbouring groups can be changed whereby the spin interactions are effectively averaged such that their contribution to the final measured value is reduced to a great extent. Since the Larmor frequency is different for each type of nucleus, an applied RF field will have a much greater effect on those spins comprising a Larmor frequency which is close to the applied frequency, than on spins of different Larmor frequency. In this manner, applied RF fields can be used in order to influence a type of nucleus while others remain unchanged.
Owing to the special problems of solid state spectroscopy, often a two-dimensional spectroscopic technique in the time domain is used in order to improve the resolution. By means of this technique, it becomes possible to investigate the interaction or "correlation" between two differing types of nuclei in a solid state--the interaction between protons and .sup.13 C nuclei is normally of great interest in many organic solids. The basic technique of the two-dimensional heteronuclear correlation in connection with solids is well known and described in many articles, as e.g. in "Heteronuclear Correlation Spectroscopy" by P. Caravatti, G. Bodenhausen and R. R. Ernst, Chemical Physics Letters Vol 89, No. 5, pp. 363-367 (July 1982) and in "Heteronuclear Correlation Spectroscopy in Rotating Solids" by P. Caravatti, L. Braunschweiler and R. R. Ernst, Chem. Phys. Letters, Vol. 100, No. 4, pp. 305-310 (September 1983). The contents of these articles are hereby incorporated by reference.
As it is described in the above-mentioned articles, the two-dimensional heteronuclear correlation technique comprises an "experiment" in the time domain which consists of four different subsequent time intervals. The first interval is called "preparation interval". During that time, one of the two examined types of nuclei is brought into an excited coherent unbalanced state which changes or "develops" during the following time intervals. The preparation interval may consist of irradiation of one single RF pulse or also of a sequence of RF pulses. The preparation interval has usually a fixed length of time.
The second time interval is called "evolution interval", during which the excited nuclear spins "develop" under the influence of the applied magnetic field, the neighbouring nuclear spins, possibly irradiating periodic RF pulse sequences and sample rotation. The evolution of the excited nuclei during this interval makes it possible to determine these frequencies. A number of "experiments" or "scans" is carried out, wherein the evolution time of the evolution interval is incremented systematically from scan to scan.
The evolution interval is followed by a "mixing interval". During the mixing interval one or more RF pulses can be applied which causes transfer of the coherence or polarization from the excited nucleus to the other investigated type of nucleus. The coherence or polarization transfer triggered by the mixing process is characteristic for the investigated nuclear system.
The mixing interval is finally followed by a "detection interval" in which the resonance frequencies of the second type of nucleus are measured. During this time interacting nuclei are usually irradiated by pulses or continuous RF energy in order to stop interaction between the two types of nuclei (decoupling).
After the Fourier transformation, the result of the multiple experiment is a two-dimensional spectral profile called heteronuclear correlation spectrum (also: 2D HETCOR PLOT). On the one axis of the plot, the detected frequencies of the second type of nucleus are displayed. The other axis represents the frequencies of the first type of nucleus detected via the repeated scans with incremented evolution times. Since the measured signal amplitude and phase of the second type of nucleus depend on the energy transfer from the originally excited first type of nucleus and the state of the first type of nucleus again depends on the evolution time, the second plot axis represents effectively the chemical shifts owing to the different first types of nuclei in a particular molecule and their spatial arrangement with respect to the second type of nucleus. The measured peaks of the plot correspond to correlations between selected nuclei of the first and second type of nucleus within a given molecule. An advantage of the heteronuclear correlation consists in that it spreads the proton resonances over the much larger area of the chemical shift of the .sup.13 C. For this reason, this technique can provide well resolved information about the chemical shift of the protons of a sample although it may not be possible to resolve said chemical shifts of the protons by means of other one-dimensional spectroscopy techniques.
It is e.g. common practice in a typical two-dimensional heteronuclear correlation experiment, which is applied to an organic material, to investigate the correlation between hydrogen nuclei (protons) .sup.1 H and .sup.13 C nuclei within the sample. To achieve this, an RF pulse is applied during the preparation interval which excites the protons. In theory, the proton spins would carry out a free precession movement during the evolution interval. During the mixing interval, the protons interact with the .sup.13 C nuclei via direct heteronuclear dipole-dipole coupling. Finally, the .sup.13 C frequencies are measured during the detection interval. One of the advantages of such an experiment is that the heteronuclear coupling between the protons and the .sup.13 C nuclei depends exclusively on the distance between the nuclei irrespective of the chemical bond. For this reason, the correlation offers the possibility to examine the stereochemistry of individual molecules and also the relative arrangement of neighbouring molecules.
The problem of this technique consists in that other couplings, like e.g. a "homonuclear" dipole-dipole coupling between protons and the "heteronuclear" dipole-dipole coupling between protons and hydrogen nuclei can cover the desired measured result if interactions during the evolution interval are permitted since they influence the measurement of chemical shifts in the proton spectrum. These last two interactions widen the peaks of the chemical shift in protons which leads to overlapping and thus prevent the assigment to the different locations. For this reason, it is necessary to suppress these two very strong interactions during the evolution interval. If a more frequent element than .sup.13 C is examined, e.g. phosphor or aluminium, it may also be necessary under certain circumstances to suppress the homonuclear interaction between these nuclei.
In general, thoroughly prepared RF pulse sequences have to be applied in order to guarantee suppression of the homonuclear and heteronuclear couplings during the evolution interval, wherein the pulses irradiate either the protons, the .sup.13 C nuclei or both simultaneously. The object of these pulse sequences consists in the suppression or averaging of the results of the undesired interactions. Many pulse sequences of this type are known in the art.
According to the prior art e.g. pulse sequences are known which suppress the homonuclear interactions between the protons in a relatively effective manner. Furthermore, other pulse sequences are known for the suppression of heteronuclear interactions between protons and .sup.13 C nuclei. In experiments for the simultaneous suppression of both the homonuclear and the heteronuclear interactions, merely the known RF pulse sequences were combined. Since, however, the known pulse sequences were not prepared with respect to a combination, very long sequences of RF pulses resulted which were necessary for suppressing both interaction types and the methods did not lead to satisfactory results. For this reason, the number of non-equivalent proton locations which could be resolved, was considerably limited. This again limited the number of connections which could be successfully examined.
The initially mentioned document EP-A 0 481 256 describes an improved method that suppresses the heteronuclear interactions more effectively. The pulse sequence proposed therein is constructed in such a manner that it can be used in connection with one of the previously known pulse sequences such that both homonuclear and heteronuclear interactions are suppressed. Moreover, the suggested pulse sequence suppresses effectively homonuclear interactions, for which reason it can be used in connection with a multitude of types of nuclei. In detail, the first type of nucleus is excited during the preparation interval by a preparation pulse and is irradiated during the evolution interval with a so-called BLEW-12 sequence (phases X Y-X-X-Y-X X Y X X-Y-X) for the homonuclear decoupling of the nuclei of the first type (i.a. protons), whereas for decoupling of the two types of nuclei (i.a. .sup.1 H-.sup.13 C) and the nuclei of the second type of nucleus (i.a. .sup.13 C--.sup.13 C), the second type of nucleus is irradiated with a pulse sequence of 12 90.degree.-RF pulses of a predetermined phase sequence, the so-called BB-12 sequence (-X Y-X X Y-X-X Y X-X Y-X). Since in this manner both the homonuclear and the heteronuclear interactions are decoupled, the protons can develop freely only under the influence of their chemical shift leading to improved resolution. After the evolution interval, two separated pulses (.theta. and .PHI. pulses) are irradiated on the protons to tilt the magnetization formed during the evolution interval into the plane perpendicular to the magnetic field for later observation. The .theta. pulse is a 90.degree. pulse and the .PHI. pulse has an angle of 63.degree. (with -Y-phase). These two pulses are followed by the so-called WIM-24 ("Windowless Isotropic Mixing) sequence which transmits nuclear polarization selectively from the protons to directly coupled carbon nuclei via the direct heteronuclear dipole interaction. The WIM-24 sequence suppresses additionally the chemical shifts of the protons and .sup.13 C nuclei and also the proton-proton and .sup.13 C--.sup.13 C homonuclear couplings, leaves, however, the proton-.sup.13 C heteronuclear coupling. The WIM-24 sequence consists of a 24 pulse sequence irradiated on the protons and a corresponding 24 pulse sequence which is simultaneously irradiated on the .sup.13 C nuclei. The sequence is prior art and is described in detail in the article "Heteronuclear Correlation Spectroscopy in Rotating Solids" by P. Caravatti, L. Braunschweiler and R. R. Ernst in Chem. Phys. Letters 100, No. 4, pp 305-310 (1983).
Finally, a continuous wave signal (CW) of a relatively high intensity is transmitted at the proton frequency during the detection interval in order to decouple the protons from the .sup.13 C nuclei in a known manner, and the .sup.13 C-FID is measured.
During the entire experiment, the solid sample is rotated in a standard manner about the "magic angle" in order to reduce broadening due to the anisotropy of the chemical shift.
In the initially mentioned document EP-A 0 481 256 (U.S. Pat. No. 5,117,186) it is also referred to the fact that instead of the WIM-24 sequence also other pulse sequences known in the art may be used in order to effect the selective cross polarization during the mixing interval and at the same time still suppress the homonuclear dipole interaction. The WIM-24 sequence was indeed preferred, but a phase- and frequency-switched Lee-Goldburg sequence (FSLG) in connection with a phase-switched .sup.13 C sequence could cause a similarly effective selective cross polarization during the mixing interval. This mixing method is described in detail in the article "Frequency-Switched Pulse Sequences: Homonuclear Decoupling and Dilute Spin NMR in Solids" by A. Bielecki, A. C. Kolbert and M. H. Levitt in Chem. Phys. Letters 155, Nos. 4,5 pp.341 (1989)
The method known from EP-A 0 481 256 (U.S. Pat. No. 5,117,186) has, however, the disadvantage, that the BLEW-12-sequence used during the evolution interval is relatively long. It is i.a. limited to about 36 microseconds to avoid RF breakthrough in the probe. This again limits the possible spin rates of the sample rotation about the magic angle since the duration of a revolution must be large compared to the time period of the BLEW-12 sequence. In practice, the spin rates are thereby limited to less than 5 kHz whereas current probes permit spin rates of over 15 kHz.
The article J.Magn.Res. A 120, p. 274-277 (1996) describes a method in which indications of resolving the chemical shift are given with a pulse sequence with high fields even without additional narrowing of the proton spectrum.
The article J.Magn.Res. A 121, p. 114-120 (1996) describes a method of imaging NMR in which the line-narrowing effect of the FSLG sequence is utilized in order to achieve slice selection.
Thus there is the need for a method of the initially mentioned kind which permits higher spin rates of the sample rotation about the magic angle.